GS28, a 28-Kilodalton Golgi SNARE That Participates in ER-Golgi Transport

Subramaniam, V. Nathan; Peter, F.; Philp, Robin; Wong, Siew Heng; Hong, Wanjin

doi:10.1126/science.272.5265.1161

Cited by 130 publications

(133 citation statements)

References 22 publications

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“…GOS 28 is a SNARE protein localized to the Golgi complex (25,27,28), where it is efficiently packaged into COPI-coated vesicles. Fab antibodies directed at GOS 28 protein partially inhibit cell-free transport and accumulate tethered, uncoated COPI transport vesicles that fail to fuse (25).…”

Section: Resultsmentioning

confidence: 99%

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

Orci

Ravazzola

Volchuk

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

103

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How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? We report immunoelectron microscopy experiments establishing that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). We also report that GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side. If so, quanta of exported proteins would transit the stack in GOS 28 -COPI vesicles via a bidirectional random walk, entering at the cis face and leaving at the trans face and percolating up and down the stack in between. Percolating vesicles carrying both post-Golgi cargo and Golgi residents up and down the stack would reconcile disparate observations on Golgi transport in cells and in cell-free systems.

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Section: Resultsmentioning

confidence: 99%

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

Orci

Ravazzola

Volchuk

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

103

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“…As shown in Figure 5B, GS32 could be detected only in untreated Golgi membranes, and its detection was completely abolished after treatment with trypsin. As a control, GS28, a SNARE anchored to the Golgi by its C-terminal membrane anchor (Subramaniam et al, 1995(Subramaniam et al, , 1996, was also not detected after treatment with trypsin. On the other hand, ST, with nearly all of its polypeptide (C-terminal) in the luminal side of the Golgi apparatus (Colley et al, 1989;Wong et al, 1992), was protected from trypsin.…”

Section: Gs32 Associates Tightly With Membranesmentioning

confidence: 99%

“…For HisX6-tagged syntaxin 6, the PCR product derived from primers A (5Ј-GCTCTCCATGGAGGACCCCTTCTTTGTAGTG-3Ј) and B (5Ј-CTCTGGATCCGCGCCGATCACTGGTCATGTGAGA-3Ј), encoding for the cytoplasmic domain of syntaxin 6, was inserted into the NcoI and BamHI sites of the pQE60 vector (Qiagen) and then transformed into E. coli M15[pREP4]. Recombinant protein was produced and purified as described previously (Subramaniam et al, 1996). For the preparation of GST fusion proteins of syntaxins 3, 4, 5, and 6 (the entire cytoplasmic domain of the syntaxins was fused to the C-terminus of GST), PCR products from primers 5 (5Ј-GGGAAT-TCTAATGAAGGACCGACTGG) and 6 (5Ј-GGCTCTAGATCATT-TCTTTCGAGCCTGACCCTG) (for syntaxin 3), primers 7 (5Ј-GG-GAATTCTAATGCGCGACAGGACCCATG) and 8 (5Ј-GGCTC-TAGATCACCTCGCCTTCTTCTGATTCTCTAG) (for syntaxin 4), primers 9 (5Ј-GGGAATTCTAATGTCCTGCCGGGATCGG) and 10 (5Ј-GGCTCTAGATCATTTGACCATGAGCCACCGATTG) (for syntaxin 5), and primers 11 (5Ј-GGGAATTCTAATGTCCATGGAGGA-CCCCTTCTTTG) and 12 (5Ј-GGCTCTAGATCAGCGCCGAT-CACTGGTCATG) (for syntaxin 6) were digested with EcoRI and XbaI and then ligated with the EcoRI/XbaI-digested pGEX-KG vector (Guan and Dixon, 1991).…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Wong

Zhang

et al. 1999

MBoC

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Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membranebound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6. INTRODUCTIONSoluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) play a central role along the secretory pathway and in other trafficking events (Graham and Emr, 1991;Pryer et al., 1992;Rothman, 1994;Rothman and Wieland, 1996;Whiteheart and Kubalek, 1995;Schekman and Orci, 1996). The action of SNAPs and NSF is mediated by SNAP receptors (SNAREs) on the membrane (Rothman, 1994;Whiteheart and Kubalek, 1995). The SNARE hypothesis is proposed to account for the specificity of vesicle transport and predicts that specific interaction between vesicle-associated SNAREs with the cognate SNAREs (t-SNAREs) on the target membrane (Rothman, 1994;Whiteheart and Kubalek, 1995) is central for vesicle docking onto and fusion with the correct target membrane. Synaptobrevin or vesicle-associated membrane proteins (VAMP 1) are vesicle-associated SNAREs associated with synaptic vesicles, whereas syntaxin 1 and the synaptosomeassociated protein of 25 kDa (SNAP-25) are t-SNAREs associated with the presynaptic membrane. The specific interaction of synaptobrevin or VAMP 1 with a complex of syntaxin 1 and SNAP-25 is involved in the docking and fusion of synaptic vesicles with the presynaptic membrane (Sö llner et al., 1993;Ferro-Novick and Jahn, 1994;Rothman, 1994;Rothman and Warren, 1994;Scheller, 1995;Sü dhof, 1995;Weber et al., 1998).In addition to involvement in synaptic vesicle docking and fusion, syntaxin-like proteins have also been implicated in transport events that occur on the plasma membrane and in intracellular organelles. In yeast, Sso1p and Sso2p are involved in the docking ‡ Corresponding author. E-m...

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“…As shown in Figure 5B, under conditions that promote SNAREcomplex assembly (lanes 2-6), substantial amounts of endobrevin were retained on the GST-␣-SNAP beads. As a positive control, GS28 (a Golgi SNARE) was similarly retained (Subramaniam et al, 1996. However, the association of endobrevin, but not GS28, with the immobilized GST-␣-SNAP is essentially abolished by NSF under conditions that promote SNARE-complex disassembly (lanes 8 -11).…”

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confidence: 98%

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

Wong

Zhang

et al. 1998

MBoC

Self Cite

108

102

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Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant ␣-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. INTRODUCTIONProtein trafficking along the exocytotic and endocytotic pathways is a vital and fundamental cellular process. Proteins destined for the exocytotic pathway are initially targeted to the endoplasmic reticulum and transported through the Golgi apparatus. At the transGolgi network (TGN), proteins are sorted to distinct post-Golgi structures such as the plasma membrane (or its subdomains) and the endosomal and lysosomal compartments (Palade, 1975;Mellman and Simons, 1992;Hong and Tang, 1993;Rothman and Wieland, 1996;Schekman and Orci, 1996). The endosomal compartment plays a central role in cellular physiology (Gruenberg and Maxfield, 1995;Mellman, 1996;Robinson et al., 1996). Endocytosed proteins are internalized from the plasma membrane via coated vesicles and then delivered to the early endosomal compartment, from which proteins can be either recycled to the plasma membrane or delivered to the late endosomal compartment and subsequently to the lysosome or the TGN.Intracellular trafficking is primarily mediated by various types of transport vesicles that bud from a donor membrane and then fuse with a specific cognate target membrane. To achieve such specificity, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis proposes that specific docking and fusion of vesicles with the cognate membrane compartment is mediated by specific interaction between the vesicle-associated SNAREs with the cognate target SNAREs on the target mem- ‡ Corresponding author. E-mail address: mcbhwj@imcb.nus. edu.sg.© 1998 by The American Society for Cell Biology 1549brane (Rothman and Warren, 1994;Bennett, 1995;Whiteheart and Kuba...

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GS28, a 28-Kilodalton Golgi SNARE That Participates in ER-Golgi Transport

Cited by 130 publications

References 22 publications

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

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